The therapeutic opioid drug methadone, that is generally used to treat cancer pain and opioid addiction, is also a potent inducer of apoptosis in human lung cancer cells, thus inhibiting their growth. This suggests a new therapeutic approach for the treatment of this cancer. However, in contrast to its central nervous system (CNS) actions, the apoptosis-inducing effects of methadone appear to be mediated through a non-opioid mechanism involving a dual- functioning bombesin receptor that is known to play a central role in the early events of pulmonary carcinogenesis. Methadone's effect is blocked in specific types of lung cancer cells that secrete high concentrations of bombesin and also by nicotine, suggesting the presence of an endogenous system coupling the apoptotic effects of nicotine, bombesin and opioids in these cells. This proposal aims to define the role of the bombesin receptor in this system, and determine whether distinct structural components are involved in the binding and functional coupling of the three ligands, with the aim of developing methadone ligands targeted towards tumor cells, and without CNS-associated toxic side effects. The specific aims of the proposal are (1) to identify the bombesin receptor type(s) involved in methadone's effects on the mitogen-activated protein (MAP) kinase signaling pathway, and apoptosis, using (a) Balb 3T3 cells transfected with the cloned bombesin receptor types (GRP, NMB and BRS-3), and (b) the different histologic types of lung cancer cells that differentially express these receptor types; (2) to determine whether nicotine acts- through this receptor type(s) to block methadone's effects; (3) to identify the structural features of the bombesin receptor involved in methadone and nicotine actions, using synthetic peptides and site-directed mutagenesis; (4) to determine the role of guanine nucleotide-binding proteins in the observed differential regulation of the bombesin receptor by methadone and bombesin. A clinical trial of methadone therapy for lung cancer patients is in progress, and these studies are expected to contribute significantly to our understanding of methadone's actions at the molecular level.